Method for producing rare fatty acid using novel enzyme, and novel rare fatty acid

ABSTRACT

The present invention provides production of hydroxylated fatty acid by a hydration reaction using a novel enzyme derived from  lactobacillus  and using fatty acid as a substrate, and further, a production method of oxo fatty acid by an enzyme reaction or chemical oxidation reaction using the hydroxylated fatty acid as a substrate. In addition, a valuable novel rare fatty acid obtained by such production method is also provided.

TECHNICAL FIELD

The present invention relates to a production method of a fatty acid.More particularly, the present invention relates to a production of ahydroxylated fatty acid by using an unsaturated fatty acid as a startingmaterial, which is characterized by a hydration reaction by a novelenzyme, and further relates to a production of an oxo fatty acid fromhydroxylated fatty acid by an enzyme reaction or chemical oxidationreaction. In addition, it relates to a novel rare fatty acid obtained bythe production method.

BACKGROUND ART

Conjugated fatty acid represented by conjugated linoleic acid (CLA) hasbeen reported to have various physiological activities such as a lipidmetabolism improving effect, an anti-arteriosclerosis action, a bodyfats decreasing action and the like (non-patent documents 1-3), and is afunctional lipid expected to be applicable to various fields ofmedicament, functional food and the like (patent documents 1, 2). WhileCLA is known to be contained in dairy products and meat products sinceit is produced by microorganisms present in the stomach of ruminant andto be present in a small amount in vegetable oil, the detailed mechanismof production thereof is not known.

The present inventors reported that enzymes present in the fungus ofLactobacillus plantarum (CLA-HY, CLA-DC, CLA-DH) are essential for thereaction to convert linoleic acid to conjugated linoleic acid (patentdocument 1). The mechanism of a series of specific reactions, thepresence of an intermediate and the like in these enzyme reactions havebeen reported (patent document 4). While these enzyme reactions wereeffective for the production of a rare fatty acid having a hydroxylgroup, a carbonyl group at the 10-position of an unsaturated fatty acidhaving 18 carbon atoms such as linoleic acid and the like, a productionmethod of a rare fatty acid having a hydroxyl group, a carbonyl group atthe 13-position has not been clarified.

In addition, it has been reported in recent years that oxo fatty acidssuch as 9-oxo-octadecadienoic acid, 13-oxo-octadecadienoic acid and thelike contained in tomato have an activity to improve lifestyle-relateddiseases, such as lipid metabolism improvement and the like (patentdocument 3, non-patent documents 4, 5). Furthermore, hydroxylated fattyacid and oxo fatty acid having a hydroxyl group, an oxo group at the10-position have been reported to have an activity to improvelifestyle-related diseases, such as metabolism improvement, lipidmetabolism improvement and the like, and an activity to improve theintestine barrier function (patent documents 5, 6), hydroxylated fattyacid and oxo fatty acid are drawing an increasing attention. However,synthesis of functional hydroxylated fatty acid, functional oxo fattyacid from unsaturated fatty acid is difficult since, in hydroxylatedfatty acid and oxo fatty acid, it is necessary to distinguish doublebonds present in a plurality in a molecule of unsaturated fatty acid andintroduce hydroxyl group, carbonyl group into particular positions.

DOCUMENT LIST Patent Documents

-   patent document 1: JP-A-2007-259712-   patent document 2: JP-A-2007-252333-   patent document 3: JP-A-2011-184411-   patent document 4: WO 2013/168310-   patent document 5: WO 2014/069227-   patent document 6: WO 2014/129384

Non-Patent Documents

-   non-patent document 1: Ha Y L, (1987), Carcinogenesis, vol. 8, no.    12, p. 1881-1887-   non-patent document 2: Clement Ip, (1991), Cancer Res., vol. 51, p.    6118-6124-   non-patent document 3: Kisun N L, (1994), Atherosclerosis, vol.    108, p. 19-25-   non-patent document 4: Kim Y-I, (2011), Mol. Nutr. Food Res., vol.    55, p. 585-593-   non-patent document 5: Kim Y-I, (2012), PLoS ONE, vol. 7, no. 2,    e31317

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method of producinghydroxylated fatty acid, oxo fatty acid by using unsaturated fatty acidas a starting material and using a novel enzyme.

Means of Solving the Problems

The present inventors have clarified a reaction wherein Lactobacillusacidophilus hydroxylates the 13-position of an unsaturated fatty acidhaving 18 carbon atoms and identified a novel enzyme (FA-HY) involved inthe production.

The present inventors have found a method of converting unsaturatedfatty acid having 16, 18, 20 carbon atoms, cis-4, cis-7, cis-10, cis-13,cis-16, cis-19-docosahexaenoic acids (DHA) and cis-9-tetradecenoic acid(myristoleic acid) to hydroxylated fatty acid by using a novel enzyme(FA-HY), and further, a method of oxidizing a hydroxyl group of theproduced substance by an enzyme reaction or chemical reaction.

The present inventor have produced found hydroxylated fatty acid and oxofatty acid having a structure not found heretofore, by a productionmethod of a new rare fatty acid by using a novel enzyme (FA-HY), andfound that they have a nuclear receptor PPARα and PPARγ agonistactivity.

To be specific, the present inventors have found that13-hydroxy-cis-9-octadecenoic acid is produced from linoleic acid byusing a novel enzyme (FA-HY), and further found that13-oxo-cis-9-octadecenoic acid is produced from13-hydroxy-cis-9-octadecenoic acid by introducing an enzyme reaction ora chemical oxidation method using chromic acid.

The present inventors have further studied and found that fatty acidshydroxylated at the 13-position, 10-position, the 15-position, the12-position are produced, using a novel enzyme (FA-HY), respectivelyfrom an unsaturated fatty acid having 18 carbon atoms and a cis doublebond at the 12-position (linoleic acid, γ-linolenic acid, α-linolenicacid, stearidonic acid etc.), unsaturated fatty acid having 16 carbonatoms and a cis double bond at the 9-position (pulmitoleic acid etc.),unsaturated fatty acid having 20 carbon atoms and a cis double bond atthe 14-position (Dihomo-γ-linolenic acid or arachidonic acid etc.),unsaturated fatty acid having 18 or 20 carbon atoms and a cis doublebond at the 11-position (cis-vaccenic acid, dihomo-γ-linolenic acid,mead acid etc.) as a substrate. Furthermore, they have found that14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid isproduced by using DHA as a substrate and 10-hydroxy-tetradecanoic acidis produced by using myristoleic acid as a substrate. In addition, theyhave found that some of the hydroxylated fatty acids in the obtainedresultant products have a structure not found before and they are novelsubstances. They have found that such hydroxylated fatty acid containinga novel fatty acid has an activity as an agonist of nuclear receptorPPARα and PPARγ, and is a substances utilizable as a metabolismimproving agent and the like.

They have also found that hydroxylated tatty acid obtained by thepresent invention as a starting material can be converted to oxo fattyacid by introducing an enzyme reaction or a chemical oxidation methodusing chromic acid. They have found a novel substance having a structurenot found before among oxo fatty acids of such resultant products. Theyhave found that such oxo fatty acid including a novel fatty acid has anactivity as an agonist of nuclear receptors PPARα and PPARγ, and theyare substances utilizable as a metabolism improving agent and the like.The present invention has been completed based on the above findings.

Accordingly, the present invention provides the following:

[1] An enzyme protein of any of the following (a)-(c):(a) the enzyme protein consisting of the amino acid sequence shown inSEQ ID NO: 2,(b) a protein comprising an amino acid sequence wherein one or pluralamino acids in the amino acid sequence shown in SEQ ID NO: 2 are deletedand/or substituted and/or inserted and/or added, and having an enzymeactivity that catalyzes a hydration reaction,(c) a protein encoded by a base sequence that hybridizes to a nucleicacid consisting of a chain sequence complementary to the base sequenceshown in SEQ ID NO: 1 under stringent conditions, and having an enzymeactivity that catalyzes a hydration reaction;[2] Lactobacillus acidophilus fungus or fungal debris thereof,comprising the enzyme protein of [1];[3] a nucleic acid encoding the enzyme protein of [1];[4] a vector comprising the nucleic acid of [3];[5] a host cell transformed with the vector of [4];[6] a method of producing the enzyme protein of [1], comprisingculturing the host cell of [5] and recovering the enzyme from theculture;[7] a method of producing a hydroxylated fatty acid having 18 carbonatoms and a hydroxyl group at the 13-position, comprising subjecting anunsaturated fatty acid having 18 carbon atoms and a cis double bond atthe 12-position to a hydration reaction using the enzyme protein of [1];[8] a method of producing an oxo fatty acid having 18 carbon atoms and acarbonyl group at the 13-position, comprising subjecting an unsaturatedfatty acid having 18 carbon atoms and a cis double bond at the12-position to a hydration reaction using the enzyme protein of [1] toinduce a hydroxylated fatty acid having 18 carbon atoms and a hydroxylgroup at the 13-position, and subjecting the hydroxylated fatty acid toa dehydrogenation reaction or chemical oxidation;[9] a method of producing a hydroxylated fatty acid having 16 carbonatoms and a hydroxyl group at the 10-position, comprising subjecting anunsaturated fatty acid having 16 carbon atoms and a cis double bond atthe 9-position to a hydration reaction using the enzyme protein of [1];[10] a method of producing an oxo fatty acid having 16 carbon atoms anda carbonyl group at the 10-position, comprising subjecting anunsaturated fatty acid having 16 carbon atoms and a cis double bond atthe 9-position to a hydration reaction using the enzyme protein of [1]to induce a hydroxylated fatty acid having 16 carbon atoms and ahydroxyl group at the 10-position, and subjecting the hydroxylated fattyacid to a dehydrogenation reaction or chemical oxidation;[11] a method of producing a hydroxylated fatty acid having 20 carbonatoms and a hydroxyl group at the 15-position, comprising subjecting anunsaturated fatty acid having 20 carbon atoms and a cis double bond atthe 14-position to a hydration reaction using the enzyme protein of [1];[12] a method of producing an oxo fatty acid having 20 carbon atoms anda carbonyl group at the 15-position, comprising subjecting anunsaturated fatty acid having 20 carbon atoms and a cis double bond atthe 14-position to a hydration reaction using the enzyme protein of [1]to induce a hydroxylated fatty acid having 20 carbon atoms and ahydroxyl group at the 15-position, and subjecting the hydroxylated fattyacid to a dehydrogenation reaction or chemical oxidation;[13] a method of producing a hydroxylated fatty acid having 18 or 20carbon atoms and a hydroxyl group at the 12-position, comprisingsubjecting an unsaturated fatty acid having 18 or 20 carbon atoms and acis double bond at the 11-position to a hydration reaction using theenzyme protein of [1];[14] a method of producing an oxo fatty acid having 18 or 20 carbonatoms and a carbonyl group at the 12-position, comprising subjecting anunsaturated fatty acid having 18 or 20 carbon atoms and a cis doublebond at the 11-position to a hydration reaction using the enzyme proteinof [1] to induce a hydroxylated fatty acid having 18 or 20 carbon atomsand a hydroxyl group at the 12-position, and subjecting the hydroxylatedfatty acid to a dehydrogenation reaction or chemical oxidation;[15] the method of [7] or [8], wherein the unsaturated fatty acid having18 carbon atoms and a cis double bond at the 12-position is linoleicacid, γ-linolenic acid, α-linolenic acid, stearidonic acid,10-hydroxy-cis-12-octadecenoic acid, 10-hydroxy-cis-6,cis-12-octadecadienoic acid, 10-hydroxy-cis-12,cis-15-octadecadienoicacid, 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid,10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoicacid, 10-oxo-cis-12,cis-15-octadecadienoic acid,10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, pinolenic acid orcolumbinic acid;[16] the method of [9] or [10], wherein the unsaturated fatty acidhaving 16 carbon atoms and a cis double bond at the 9-position ispulmitoleic acid;[17] the method of [11] or [12], wherein the unsaturated fatty acidhaving 20 carbon atoms and a cis double bond at the 14-position iscis-11,cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoicacid, dihomo-γ-linolenic acid,cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, arachidonic acid,sciadonic acid or juniperonic acid;[18] the method of [13] or [14], wherein the unsaturated fatty acidhaving 18 or 20 carbon atoms and a cis double bond at the 11-position iscis-vaccenic acid, cis-11-cis-14-eicosadienoic acid,cis-11,cis-14,cis-17-eicosatrienoic, dihomo-γ-linolenic acid, mead acid,cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid or arachidonic acid;[19] a method of producing14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid,comprising subjectingcis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to ahydration reaction using the enzyme protein of [1];[20] a method of producing14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid,comprising subjectingcis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to ahydration reaction using the enzyme protein of [1] to induce14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, andsubjecting the hydroxylated fatty acid to a dehydrogenation reaction orchemical oxidation;[21] a method of producing 10-hydroxy-tetradecanoic acid, comprisingsubjecting cis-9-tetradecenoic acid (myristoleic acid) to a hydrationreaction using the enzyme protein of [1];[22] a method of producing 10-oxo-tetradecanoic acid, comprisingsubjecting cis-9-tetradecenoic acid (myristoleic acid) to a hydrationreaction using the enzyme protein of [1] to induce10-hydroxy-tetradecanoic acid, and subjecting the hydroxylated fattyacid to a dehydrogenation reaction or chemical oxidation;[23] the method of [8], [10], [12], [14]-[18], [20] or [22], wherein thedehydrogenation reaction uses a dehydrogenase derived fromlactobacillus;[24] the method of [23], wherein the lactobacillus is Lactobacillusplantarum PERM BP-10549 strain;[25] 13-hydroxy-cis-9,cis-15-octadecadienoic acid,13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid,10,13-dihydroxy-cis-6-octadecenoic acid,10,13-dihydroxy-cis-15-octadecenoic acid,10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid,10-oxo-13-hydroxy-cis-6-octadecenoic acid,10-oxo-13-hydroxy-cis-15-octadecenoic acid, 10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid, 13-hydroxy-cis-5, cis-9-octadecadienoicacid or 13-hydroxy-trans-5,cis-9-octadecadienoic acid, which is producedby the method of [7];[26] 15-hydroxy-cis-11,cis-17-eicosadienoic acid,15-hydroxy-cis-8,cis-11-eicosadienoic acid,15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid,15-hydroxy-cis-5,cis-11-eicosadienoic acid or15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced bythe method of [11];[27] 12-hydroxy-cis-14-eicosenoic acid,12-hydroxy-cis-14,cis-17-eicosadienoic acid or12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid, which is produced bythe method of [13];[28] 13-oxo-cis-6,cis-9-octadecadienoic acid,13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid,10,13-dioxo-cis-6-octadecenoic acid, 10,13-dioxo-cis-15-octadecenoicacid, 10,13-dioxo-cis-6,cis-15-octadecadienoic acid,13-oxo-cis-5,cis-9-octadecadienoic acid or13-oxo-trans-5,cis-9-octadecadienoic acid, which is produced by themethod of [8];[29] 15-oxo-cis-11,cis-17-eicosadienoic acid,15-oxo-cis-8,cis-11-eicosadienoic acid,15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid,15-oxo-cis-5,cis-11-eicosadienoic acid or15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced by themethod of [12];[30] 12-oxo-cis-14-eicosenoic acid, 12-oxo-cis-14,cis-17-eicosadienoicacid, 12-oxo-cis-8,cis-14-eicosadienoic acid,12-oxo-cis-5,cis-8-eicosadienoic acid or12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid, which is produced by themethod of [14];[31] 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acidproduced by the method of [19];[32] 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid,which is produced by the method of [20];[33] a fatty acid-containing substance comprising the hydroxylated fattyacid or oxo fatty acid of any one of [25]-[32];[34] use of Lactobacillus acidophilus fungus or fungal debris thereof,comprising the enzyme protein of [1] in the production of thehydroxylated fatty acid or oxo fatty acid of any one of [25]-[32].

Effect of the Invention

In the present invention, a fatty acid hydration enzyme (FA-HY)conventionally not known was found, a method of converting unsaturatedfatty acid having 16, 18 or 20 carbon atoms, DHA or myristoleic acid toa hydroxylated fatty acid was found, and further, a method of oxidizinga hydroxyl group of the resulting substance by an enzyme reaction or achemical reaction was found. The rare fatty acid etc. to be produced areextremely useful since they are used in various fields such asmedicament, food, cosmetic and the like. In addition, a novel rare fattyacid can be produced by the production using a novel enzyme, and thenovel rare fatty acid also has an activity as an agonist of nuclearreceptors PPARα and PPARγ. Therefore, it can be a useful substance invarious fields such as medicament, food, cosmetic and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of PPARα agonist activity of rare fatty acidsderived from linoleic acid, α-linolenic acid, γ-linolenic acid, andarachidonic acid. EtOH shows a negative control (ethanol addition), andGW7647 shows a positive control (PPARα agonist addition). The verticalaxis shows relative luciferase activity.

FIG. 2 shows the results of PPARγ agonist activity of rare fatty acidsderived from linoleic acid, α-linolenic acid, γ-linolenic acid, andarachidonic acid. EtOH shows a negative control (ethanol addition), andTro shows a positive control (PPARγ agonist addition). The vertical axisshows relative luciferase activity.

DESCRIPTION OF EMBODIMENTS

The present invention is explained in detail below.

the present invention provides a novel fatty acid hydration enzyme“FA-HY”.

Specifically, the novel enzyme “FA-HY” of the present invention is

(a) the enzyme protein consisting of the amino acid sequence shown inSEQ ID NO: 2,(b) a protein comprising an amino acid sequence wherein one or pluralamino acids in the amino acid sequence shown in SEQ ID NO: 2 are deletedand/or substituted and/or inserted and/or added, and having an enzymeactivity that the enzyme protein consisting of the amino acid sequenceshown in SEQ ID NO: 2 has, or(c) a protein encoded by a base sequence that hybridizes to a nucleicacid consisting of a chain sequence complementary to the base sequenceshown in SEQ ID NO: 1 under stringent conditions, and having an enzymeactivity that the enzyme protein consisting of the amino acid sequenceshown in SEQ ID NO: 2 has.

More specific examples of the above-mentioned (b) include a proteincontaining (i) an amino acid sequence which is the amino acid sequenceshown in SEQ ID NO: 2, wherein 1-20, preferably 1-10, more preferably1-several (5, 4, 3 or 2) amino acids are deleted, (ii) an amino acidsequence which is the amino acid sequence shown in SEQ ID NO: 2, wherein1-20, preferably 1-10, more preferably 1-several number (5, 4, 3 or 2)amino acids are added, (iii) an amino acid sequence which is the aminoacid sequence shown in SEQ ID NO: 2, wherein 1-20, preferably 1-10, morepreferably 1-several (5, 4, 3 or 2) amino acids are inserted, (iv) anamino acid sequence which is the amino acid sequence shown in SEQ ID NO:2, wherein 1-20, preferably 1-10, more preferably 1-several (5, 4, 3 or2) amino acids are substituted by other amino acids, or (v) an aminoacid sequence obtained by combining them. When amino acids with similarproperties (e.g., glycine and alanine, valine and leucine andisoleucine, serine and threonine, aspartic acid and glutamic acid,asparagine and glutamine, lysine and arginine, cysteine and methionine,phenylalanine and tyrosine etc.) are substituted with each other and thelike, a greater number of substitutions and the like are possible.

When amino acids are deleted, substituted or inserted as mentionedabove, the positions of deletion, substitution and insertion are notparticularly limited as long as the above-mentioned enzyme activity ismaintained.

In the above-mentioned (c), the “stringent conditions” are conditionsunder which nucleotide sequences having high identity, for example,identity of 70, 80, 90, 95 or 99% or above, hybridize to each other andnucleotide sequences having identity lower than that do not hybridize;specifically, conditions of washing once, more preferably 2-3 times, atthe salt concentration and temperature corresponding to those in thewashing conditions of general Southern hybridization (60° C., 1×SSC,0.1% SDS, preferably, 0.1×SSC, 0.1% SDS, more preferably. 68° C.,0.1×SSC, 0.1% SDS) and the like.

Regarding the above-mentioned (b) or (c), the enzyme activity that theenzyme protein consisting of the amino acid sequence shown in SEQ ID NO:2 has is not particularly limited as long as it has at least one,preferably all, of (1) an enzyme activity capable of converting anunsaturated fatty acid having 18 carbon atoms and a cis double bond atthe 12-position (hereinafter sometimes to be abbreviated as “cis-12unsaturated fatty acid”) utilized as a substrate to a hydroxylated fattyacid having 18 carbon atoms and a hydroxyl group at the 13-position(hereinafter sometimes to be abbreviated as “13-hydroxy fatty acid”)(reaction 1), (2) an enzyme activity capable of converting anunsaturated fatty acid having 16 carbon atoms and a cis double bond atthe 9-position (hereinafter sometimes to be abbreviated as “cis-9unsaturated fatty acid”)) utilized as a substrate to a hydroxylatedfatty acid having 16 carbon atoms and a hydroxyl group at the10-position (hereinafter sometimes to be abbreviated as “10-hydroxyfatty acid”) (reaction 2), (3) an enzyme activity capable of convertingan unsaturated fatty acid having 20 carbon atoms and a cis double bondat the 14-position (hereinafter sometimes to be abbreviated as “cis-14unsaturated fatty acid”)) utilized as a substrate to a hydroxylatedfatty acid having 20 carbon atoms and a hydroxyl group at the15-position (hereinafter sometimes to be abbreviated as “15-hydroxyfatty acid”) (reaction 3), (4) an enzyme activity capable of convertingan unsaturated fatty acid having 18 or 20 carbon atoms and a cis doublebond at the 11-position (hereinafter sometimes to be abbreviated as“cis-11 unsaturated fatty acid”)) utilized as a substrate to ahydroxylated fatty acid having 18 or 20 carbon atoms and a hydroxylgroup at the 12-position (hereinafter sometimes to be abbreviated as“12-hydroxy fatty acid”) (reaction 4), an enzyme activity capable ofconverting cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid(DHA) to 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoicacid (reaction A), and an enzyme activity capable of convertingcis-9-tetradecenoic acid (myristoleic acid) to 10-hydroxy-tetradecanoicacid (reaction I)

The above-mentioned “cis-12 unsaturated fatty acid”, “cis-9 unsaturatedfatty acid”, “cis-14 unsaturated fatty acid”, and “cis-11 unsaturatedfatty acid” are not particularly limited as long as they are anunsaturated fatty acid having 18 carbon atoms and a cis double bond atthe 12-position, an unsaturated fatty acid having 16 carbon atoms and acis double bond at the 9-position, an unsaturated fatty acid having 20carbon atoms and a cis double bond at the 14-position, an unsaturatedfatty acid having 18 or 20 carbon atoms and a cis double bond at the11-position, respectively and, for example, monovalent unsaturated fattyacid, divalent unsaturated fatty acid, trivalent unsaturated fatty acid,tetravalent unsaturated fatty acid, pentavalent unsaturated fatty acidand the like can be mentioned. In the present specification, the “fattyacid” encompasses not only free acids but also ester form, salt withbasic compound and the like. The “DHA” and “myristoleic acid” alsoencompass not only free acids but also ester form, salt with basiccompound and the like.

The FA-HY of the present invention can be isolated from, for example,the fungus, culture medium of Lactobacillus acidophilus by a proteinseparation and purification technique known per se. Alternatively, FA-HYmay be used as the fungus of Lactobacillus acidophilus containing FA-HYor fungal debris thereof. The fungus of Lactobacillus acidophiluscontaining FA-HY is not particularly limited as long as it contains theFA-HY of the present invention and, for example, NITE BP-01788 and thelike can be mentioned. Alternatively, FA-HY can also be produced as arecombinant protein by isolating a gene encoding FA-HY according to themethod described in Example 2, subcloning same into a suitable vector,introducing same into a suitable host such as Escherichia coli and thelike and culturing same. FA-HY may be a purified one or a crudelypurified one. Alternatively, hydratase may be expressed in fungus suchas Escherichia coli and the like and the fungus itself may be used orculture medium thereof may be used. Furthermore, the enzyme may be of afree form, or immobilized by various carriers.

As a vector containing a nucleic acid encoding FA-HY of the presentinvention, one suitable for a host cell to be introduced with the vectormay be appropriately selected according to the object (e.g., proteinexpression) and can be used. In the case of an expression vector, itcontains the nucleic acid of the present invention, which is operablylinked to an appropriate promoter, and preferably contains atranscription termination signal, i.e., terminator region, at thedownstream of the nucleic acid of the present invention. Furthermore, itcan also contain a selection marker gene for selection of a transformant(drug resistance gene, gene that complements auxotrophic mutation etc.).Also, it may contain a sequence encoding a tag sequence useful forseparation and purification of the expressed protein and the like. Inaddition, the vector may be incorporated into the genome of a targethost cell. The vector of the present invention can be introduced into atarget host cell by a transformation method known per se such as acompetent cell method, a protoplast method, a calcium phosphatecoprecipitation method and the like.

In the present invention, the “host cell” may be any cell as long as itcan express a vector containing a nucleic acid encoding EA-HY of thepresent invention, and bacterium, yeast, fungi, higher eukaryotic celland the like can be mentioned. Examples of the bacterium includegram-positive bacteria such as bacillus, Streptomyces and the like andgram negative bacteria such as Escherichia coli and the like. Arecombinant cell introduced with a vector containing a nucleic acidencoding FA-HY can be cultivated by a method known per se which issuitable for the host cell.

“Purification” of the FA-HY of the present invention can be performed bya method known per se, for example, fungi collected by centrifugationand the like are ruptured by ultrasonication or glass beads and thelike, solid such as cell debris is removed by centrifugation and thelike, and the like to give a crude enzyme solution, which is subjectedto a salting out method using ammonium sulfate, sodium sulfate and thelike, chromatographys such as ion exchange chromatography, gelfiltration chromatography, affinity chromatography and the like, gelelectrophoresis and the like.

The FA-HY of the present invention has, as mentioned above, an enzymeactivity capable of converting cis-12 unsaturated fatty acid, cis-9unsaturated fatty acid, cis-14 unsaturated fatty acid, cis-11unsaturated fatty acid, DHA, myristoleic acid utilized as substrates to13-hydroxy fatty acid, 10-hydroxy fatty acid, 15-hydroxy fatty acid,12-hydroxy fatty acid,14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid,10-hydroxy-tetradecanoic acid, respectively. Therefore, the presentinvention also provides [1] a method of producing 13-hydroxy fatty acidfrom cis-12 unsaturated fatty acid by a hydration reaction using theFA-HY of the present invention (production method 1), [2] a method ofproducing 10-hydroxy fatty acid from cis-9 unsaturated fatty acid by ahydration reaction using the FA-HY of the present invention (productionmethod 2), [3] a method of producing 15-hydroxy fatty acid from cis-14unsaturated fatty acid by a hydration reaction using the FA-HY of thepresent invention (production method 3), [4] a method of producing12-hydroxy fatty acid from cis-11 unsaturated fatty acid by a hydrationreaction using the FA-HY of the present invention (production method 4),[A] a method of producing14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid fromDHA by a hydration reaction using the FA-HY of the present invention(production method A), and [I] a method of producing10-hydroxy-tetradecanoic acid from myristoleic acid by a hydrationreaction using the FA-HY of the present invention (production method I).

Examples of the “cis-12 unsaturated fatty acid” in the production method1 of the present invention include cis-9,cis-12-octadecadienoic acid(linoleic acid), cis-6,cis-9,cis-12-octadecatrienoic acid (γ-linolenicacid), cis-9,cis-12,cis-15-octadecatrienoic acid (α-linolenic acid),cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (stearidonic acid), aswell as 10-hydroxy-cis-12-octadecenoic acid,10-hydroxy-cis-6,cis-12-octadecadienoic acid,10-hydroxy-cis-12,cis-15-octadecadienoic acid,10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid,10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoicacid, 10-oxo-cis-12,cis-15-octadecadienoic acid,10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid,cis-5,cis-9,cis-12-octadecatrienoic acid(pinolenic acid),trans-5,cis-9,cis-12-octadecatrienoic acid (columbinic acid), which arenow producible by WO 2013/168310, and the like. These substrates may beobtained by a method other than WO 2013/168310.

Examples of the “13-hydroxy fatty acid” produced by production method 1of the present invention include 13-hydroxy-cis-9-octadecenoic acidinduced from cis-9,cis-12-octadecadienoic acid (linoleic acid),13-hydroxy-cis-6,cis-9-octadecadienoic acid induced fromcis-6,cis-9,cis-12-octadecatrienoic acid (γ-linolenic acid),13-hydroxy-cis-9,cis-15-octadecadienoic acid induced fromcis-9,cis-12,cis-15-octadecatrienoic acid (α-linolenic acid),13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid induced fromcis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (stearidonic acid),10,13-dihydroxy-octadecanoic acid induced from10-hydroxy-cis-12-octadecenoic acid, 10,13-dihydroxy-cis-6-octadecenoicacid induced from 10-hydroxy-cis-6,cis-12-octadecadienoic acid,10,13-dihydroxy-cis-15-octadecenoic acid induced from 10-hydroxy-cis-12,cis-15-octadecadienoic acid,10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid induced from10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid,10-oxo-13-hydroxy-octadecanoic acid induced from10-oxo-cis-12-octadecenoic acid, 10-oxo-13-hydroxy-cis-6-octadecenoicacid induced from 10-oxo-cis-6, cis-12-octadecadienoic acid,10-oxo-13-hydroxy-cis-15-octadecenoic acid induced from10-oxo-cis-12,cis-15-octadecadienoic acid,10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid induced from10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, 13-hydroxy-cis-5,cis-9-octadecadienoic acid induced fromcis-5,cis-9,cis-12-octadecatrienoic acid (pinolenic acid),13-hydroxy-trans-5, cis-9-octadecadienoic acid induced fromtrans-5,cis-9,cis-12-octadecatrienoic acid (columbinic acid) and thelike.

Examples of the “cis-9 unsaturated fatty acid” in production method 2 ofthe present invention include cis-9-hexadecenoic acid (pulmitoleic acid)and the like.

Examples of the “10-hydroxy fatty acid” produced by production method 2of the present invention include 10-hydroxy-hexadecanoic acid inducedfrom cis-9-hexadecenoic acid (pulmitoleic acid) and the like.

Examples of the “cis-14 unsaturated fatty acid” in production method 3of the present invention include cis-11,cis-14-eicosadienoic acid,cis-11,cis-14,cis-17-eicosatrienoic acid,cis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid),cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid,cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid),cis-5,cis-11,cis-14-eicosatrienoic acid (sciadonic acid),cis-5,cis-11,cis-14,cis-17-eicosatetraenoic acid (juniperonic acid) andthe like.

Examples of the “15-hydroxy fatty acid” produced by production method 3of the present invention include 15-hydroxy-cis-11-eicosenoic acidinduced from cis-11,cis-14-eicosadienoic acid,15-hydroxy-cis-11,cis-17-eicosadienoic acid induced fromcis-11,cis-14,cis-17-eicosatrienoic acid,15-hydroxy-cis-8,cis-11-eicosadienoic acid induced fromcis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid),15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid induced fromcis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid,15-hydroxy-cis-5,cis-8,cis-11-eicosatrienoic acid induced fromcis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid),15-hydroxy-cis-5,cis-11-eicosadienoic acid induced fromcis-5,cis-11,cis-14-eicosatrienoic acid (sciadonic acid),15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid induced fromcis-5,cis-11,cis-14,cis-17-eicosatetraenoic acid (juniperonic acid) andthe like.

Examples of the “cis-11 unsaturated fatty acid” in production method 4of the present invention include cis-11-octadecenoic acid (cis-vaccenicacid), cis-11,cis-14-eicosadienoic acid,cis-11,cis-14,cis-17-eicosatrienoic acid,cis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid),cis-5,cis-8,cis-11-eicosatrienoic acid (mead acid),cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid,cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid) andthe like.

Examples of the “12-hydroxy fatty acid” produced by production method 4of the present invention include 12-hydroxy-octadecanoic acid inducedfrom cis-11-octadecenoic acid(cis-vaccenic acid),12-hydroxy-cis-14-eicosenoic acid induced fromcis-11,cis-14-eicosadienoic acid, 12-hydroxy-cis-14,cis-17-eicosadienoicacid induced from cis-11,cis-14,cis-17-eicosatrienoic acid,12-hydroxy-cis-8,cis-14-eicosadienoic acid induced fromcis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid),12-hydroxy-cis-5,cis-8-eicosadienoic acid induced fromcis-5,cis-8,cis-11-eicosatrienoic acid (mead acid),12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid induced fromcis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid,12-hydroxy-cis-5,cis-8,cis-14-eicosatrienoic acid induced fromcis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid) andthe like.

The hydration reaction may be performed in a suitable buffer (e.g.,phosphate buffer, tris buffer, borate buffer etc.) by mixing unsaturatedfatty acid, which is a substrate, and FA-HY of the present invention atsuitable concentrations and incubating the mixture. The substrateconcentration is, for example, 1-1000 g/L, preferably 10-500 g/L, morepreferably 20-250 g/L. The amount of the aforementioned FA-HY to beadded is, for example, 0.001-10 mg/mL, preferably 0.1-5 mg/mL, morepreferably 0.2-2 mg/mL.

A “cofactor” may be used for a hydration reaction (reaction 1-4,reaction A or reaction I) and, for example, FAD and the like can beused. The concentration of addition may be any as long as the hydrationreaction proceeds efficiently. It is preferably 0.001-20 mM, morepreferably 0.01-10 mM.

Furthermore, an “activator” may be used for the hydration reaction and,for example, 1 or 2 compounds selected from the group consisting of NADHand NADPH can be mentioned. The concentration of addition thereof may beany as long as the hydration reaction proceeds efficiently. It ispreferably 0.1-20 mM, more preferably 1-10 mM.

The hydration reaction is desirably performed at a preferabletemperature and in a preferable pH range for the FA-HY of the presentinvention. For example, the reaction temperature is 5-50° C., preferably20-45° C. The pH of the reaction mixture is, for example, pH 4-10,preferably pH 5-9. The reaction time is not particularly limited and itis, for example, 10 min-72 hr, preferably 30 min-36 hr.

In one preferable embodiment of the present invention, the FA-HY of thepresent invention is provided to the reaction system in the form ofrecombinant cells (e.g., Escherichia coli, Bacillus subtilis, yeast,insect cell, animal cell etc.) introduced with an expression vectorcontaining a nucleic acid encoding same. In this case, the hydrationreaction can also be performed by cultivating the cells in a liquidmedium suitable for the culture of the cells and added with cofactor anda substrate and, where necessary, an activator.

Furthermore, by a dehydrogenation reaction or chemical oxidation usingchrome acid, an oxo fatty acid having 18 carbon atoms and a carbonylgroup at the 13-position (hereinafter sometimes to be abbreviated as“13-oxo fatty acid”) is produced from 13-hydroxy fatty acid obtained inproduction methods 1-4, production method A, production method I of thepresent invention (reaction 5), an oxo fatty acid having 16 carbon atomsand a carbonyl group at the 10-position (hereinafter sometimes to beabbreviated as “10-oxo fatty acid”) is produced from 10-hydroxy fattyacid (reaction 6), an oxo fatty acid having 20 carbon atoms and acarbonyl group at the 15-position (hereinafter sometimes to beabbreviated as “15-oxo fatty acid”) is produced from 15-hydroxy fattyacid (reaction 7), an oxo fatty acid having 18 or 20 carbon atoms and acarbonyl group at the 12-position (hereinafter sometimes to beabbreviated as “12-oxo fatty acid”) is produced from 12-hydroxy fattyacid (reaction 8),14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid isproduced from14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid(reaction B), and 10-oxo-tetradecanoic acid is produced from10-hydroxy-tetradecanoic acid (reaction II).

Therefore, the present invention also provides [5] a method of producing13-oxo fatty acid, comprising subjecting cis-12 unsaturated fatty acidto a hydration reaction using the FA-HY of the present invention toinduce 13-hydroxy fatty acid, and subjecting the 13-hydroxy fatty acidto a dehydrogenation reaction or chemical oxidation (production method5), [6] a method of producing 10-oxo fatty acid, comprising subjectingcis-9 unsaturated fatty acid to a hydration reaction using the FA-HY ofthe present invention to induce 10-hydroxy fatty acid, and subjectingthe 10-hydroxy fatty acid to a dehydrogenation reaction or chemicaloxidation (production method 6), [7] a method of producing 15-oxo fattyacid, comprising subjecting cis-14 unsaturated fatty acid to a hydrationreaction using the FA-HY of the present invention to induce 15-hydroxyfatty acid, and subjecting the 15-hydroxy fatty acid to adehydrogenation reaction or chemical oxidation (production method 7),[8] a method of producing 12-oxo fatty acid, comprising subjectingcis-11 unsaturated fatty acid to a hydration reaction using the FA-HY ofthe present invention to induce 12-hydroxy fatty acid, and subjectingthe 12-hydroxy fatty acid to a dehydrogenation reaction or chemicaloxidation (production method 8), [B] a method of producing14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid,comprising subjecting DHA to a hydration reaction using the EA-HY of thepresent invention to induce14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, andsubjecting the14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid to adehydrogenation reaction or chemical oxidation (production method B),and [II] a method of producing 10-oxo-tetradecanoic acid, comprisingsubjecting myristoleic acid to a hydration reaction using the FA-HY ofthe present invention to induce 10-hydroxy-tetradecanoic acid, andsubjecting the 10-hydroxy-tetradecanoic acid to a dehydrogenationreaction or chemical oxidation (production method II).

The “cis-12 unsaturated fatty acid”, “cis-9 unsaturated fatty acid”,“cis-14 unsaturated fatty acid”, “cis-11 unsaturated fatty acid” in theproduction methods 5-8 of the present invention are the same as thesubstrates in the above-mentioned production methods 1-4.

Examples of the “13-oxo fatty acid” produced by the production method 5of the present invention include 13-oxo-cis-9-octadecenoic acid inducedfrom cis-9,cis-12-octadecadienoic acid (linoleic acid),13-oxo-cis-6,cis-9-octadecadienoic acid induced fromcis-6,cis-9,cis-12-octadecatrienoic acid (γ-linolenic acid),13-oxo-cis-9,cis-15-octadecadienoic acid induced fromcis-9,cis-12,cis-15-octadecatrienoic acid (α-linolenic acid),13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid induced fromcis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (stearidonic acid),10,13-dioxo-octadecanoic acid induced from10-hydroxy-cis-12-octadecenoic acid or 10-oxo-cis-12-octadecenoic acid,10,13-dioxo-cis-6-octadecenoic acid induced from10-hydroxy-cis-6,cis-12-octadecadienoic acid or10-oxo-cis-6,cis-12-octadecadienoic acid,10,13-dioxo-cis-15-octadecenoic acid induced from10-hydroxy-cis-12,cis-15-octadecadienoic acid or10-oxo-cis-12,cis-15-octadecadienoic acid,10,13-dioxo-cis-6,cis-15-octadecadienoic acid induced from10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid or10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid,13-oxo-cis-5,cis-9-octadecadienoic acid induced fromcis-5,cis-9,cis-12-octadecatrienoic acid (pinolenic acid),13-oxo-trans-5,cis-9-octadecadienoic acid induced fromtrans-5,cis-9,cis-12-octadecatrienoic acid (columbinic acid) and thelike.

Examples of the “10-oxo fatty acid” produced by the production method 6of the present invention include 10-oxo-hexadecanoic acid induced fromcis-9-hexadecenoic acid (pulmitoleic acid) and the like.

Examples of the “15-oxo fatty acid” produced by the production method 7of the present invention include 15-oxo-cis-11-eicosenoic acid inducedfrom cis-11,cis-14-eicosadienoic acid,15-oxo-cis-11,cis-17-eicosadienoic acid induced fromcis-11,cis-14,cis-17-eicosatrienoic acid,15-oxo-cis-8,cis-11-eicosadienoic acid induced fromcis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid),15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid induced fromcis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid,15-oxo-cis-5,cis-8,cis-11-eicosatrienoic acid induced fromcis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid),15-oxo-cis-5,cis-11-eicosadienoic acid induced fromcis-5,cis-11,cis-14-eicosatrienoic acid (sciadonic acid),15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid induced fromcis-5,cis-11,cis-14,cis-17-eicosatetraenoic acid (juniperonic acid) andthe like.

Examples of the “12-oxo fatty acid” produced by the production method 8of the present invention include 12-oxo-octadecanoic acid induced fromcis-11-octadecenoic acid (cis-vaccenic acid), 12-oxo-cis-14-eicosenoicacid induced from cis-11,cis-14-eicosadienoic acid,12-oxo-cis-14,cis-17-eicosadienoic acid induced fromcis-11,cis-14,cis-17-eicosatrienoic acid, 12-oxo-cis-8,cis-14-eicosadienoic acid induced fromcis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid),12-oxo-cis-5,cis-8-eicosadienoic acid induced fromcis-5,cis-8,cis-11-eicosatrienoic acid (mead acid),12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid induced fromcis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid,12-oxo-cis-5,cis-8,cis-14-eicosatrienoic acid induced fromcis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid) andthe like.

The dehydrogenase to be used in the production methods 5-8, productionmethod B or production method II of the present invention is notparticularly limited as long as it is an enzyme capable of converting13-hydroxy fatty acid, 10-hydroxy fatty acid, 15-hydroxy fatty acid,12-hydroxy fatty acid,14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid,10-hydroxy-tetradecanoic acid utilized as substrates to 13-oxo fattyacid, 10-oxo fatty acid, 15-oxo fatty acid, 12-oxo fatty acid,14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid,10-oxo-tetradecanoic acid, respectively and, for example,lactobacillus-derived hydroxylated fatty acid-dehydrogenase (CLA-DH) ispreferable. More preferred is Lactobacillus plantarum-derived CLA-DH,and particularly preferred is L. plantarum FERN BP-10549 strain-derivedCLA-DH. CLA-DH can be obtained by the method described inJP-A-2007-259712, the method described in WO 2013/168310. Dehydrogenasemay be a purified one or a crudely purified one. Alternatively,dehydrogenase may be expressed in fungus such as Escherichia coli andthe like and the fungus itself may be used or culture medium thereof maybe used. Furthermore, the enzyme may be of a free form, or immobilizedby various carriers.

The dehydrogenation reaction is performed in a suitable buffer (e.g.,phosphate buffer, tris buffer, borate buffer etc.) by mixing 13-hydroxyfatty acid, 10-hydroxy fatty acid, 15-hydroxy fatty acid, 12-hydroxyfatty acid, 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoicacid, 10-hydroxy-tetradecanoic acid as substrates and dehydrogenase atsuitable concentrations and incubating the mixture. The substrateconcentration is, for example, 0.01-100 g/L, preferably 0.05-50 g/L,more preferably 0.1-5 g/L. The amount of dehydrogenase to be added is,for example, 0.001-10 mg/mL, preferably 0.005-1 mg/mL, more preferably0.05-0.2 mg/mL.

A “cofactor” may be used for the dehydrogenation reaction and, forexample, NAD⁺, NADP⁺ and the like can be used. The concentration ofaddition thereof may be any as long as the hydration reaction proceedsefficiently. It is preferably 0.001-20 mM, more preferably 0.01-10 mM.

The dehydrogenation reaction is desirably performed within the ranges ofpreferable temperature and preferable pH of dehydrogenase. For example,the reaction temperature is 5-50° C., preferably 20-45° C. The pH of thereaction mixture is, for example, pH 4-10, preferably pH 5-9. Thereaction time is not particularly limited and it is, for example, 10min-72 hr, preferably 30 min-36 hr.

In one embodiment of the present invention, dehydrogenase is subjectedto the reaction system in the form of recombinant cells (e.g.,Escherichia coli, Bacillus subtilis, yeast, insect cell, animal celletc.) introduced with an expression vector containing a nucleic acidencoding same. In this case, the oxidation reaction can also beperformed by cultivating the cells in a liquid medium suitable for theculture of the cells and added with a substrate and, where necessary, acofactor and an activator.

In addition, by replacing the dehydrogenation reaction with a chemicaloxidation using chromic acid, an oxo fatty acid similar to that byenzyme reaction can be chemically obtained.

As the chemical oxidation, methods known per se, for example, chromicacid oxidation, preferably Jones oxidation and the like can bementioned. As the chromic acid, salts or complexes of the compound suchas anhydrous chromic acid CrO₃, chromic acid H₂CrO₄ and dichromic acidH₂Cr₂O₇ can be used.

To be specific, sulfuric acid (2.3 ml) and water (7.7 ml) are added toanhydrous chromic acid (2.67 g), and acetone (90 ml) is added to themixture to give a chromic acid solution. 2 g of hydroxylated fatty acidand 40 ml of acetone are added in an Erlenmeyer flask, and theabove-mentioned chromic acid solution is added by one drop whilestirring in a stirrer on ice. When the solution turns from blue to teagreen, dropwise addition of the chromic acid solution is stopped, andthe reaction is discontinued with isopropyl alcohol. The precipitatedsediment is filtered through filter paper, placed in a separatingfunnel, diethyl ether (150 ml) and Milli-Q water (300 ml) are added, themixture is shaken well, and the diethyl ether layer is washed severaltimes with Milli-Q water. To the diethyl ether layer after washing isadded an appropriate amount of sodium sulfate (anhydrous), the mixtureis stirred and the residual water is removed. The anhydrous sodiumsulfate added is filtered off through filter paper, the obtained diethylether layer is concentrated by a rotary evaporator, and the reactionproduct (oxo fatty acid) and unreacted substrate are extracted.

An extract obtained by an oxidation reaction with anhydrous chromic acid(mixture containing substrate and resultant product (oxo fatty acid)) issubjected to moderate-pressure chromatography, a solution that comes outfrom the column is recovered in fractions. The recovered each fractionis analyzed by LC/MS and gas chromatography, fractions containing oxofatty acid alone are collected and concentrated by a rotary evaporator.A part of the obtained final resultant product is methylesterified, thepurity of oxo fatty acid is evaluated by gas chromatography, and oxofatty acid having a purity of not less than 98% can be obtained.

The following hydroxylated fatty acid and oxo fatty acid obtained by theproduction methods 1-8 and production methods A, B of the presentinvention are novel fatty acids having structures conventionally notknown.

<Hydroxylated Fatty Acid>

-   13-hydroxy-cis-9, cis-15-octadecadienoic acid-   13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid-   13-hydroxy-cis-5, cis-9-octadecadienoic acid-   13-hydroxy-trans-5, cis-9-octadecadienoic acid-   12-hydroxy-cis-14-eicosenoic acid-   12-hydroxy-cis-14,cis-17-eicosadienoic acid-   15-hydroxy-cis-11,cis-17-eicosadienoic acid-   15-hydroxy-cis-8,cis-11-eicosadienoic acid-   12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid-   15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid-   15-hydroxy-cis-5,cis-11-eicosadienoic acid-   15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid-   10,13-dihydroxy-cis-6-octadecenoic acid-   10,13-dihydroxy-cis-15-octadecenoic acid-   10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid-   10-oxo-13-hydroxy-cis-6-octadecenoic acid-   10-oxo-13-hydroxy-cis-15-octadecenoic acid-   10-oxo-13-hydroxy-cis-6, cis-15-octadecadienoic acid-   14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid

<Oxo Fatty Acid>

-   13-oxo-cis-6, cis-9-octadecadienoic acid-   13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid-   13-oxo-cis-5, cis-9-octadecadienoic acid-   13-oxo-trans-5, cis-9-octadecadienoic acid-   12-oxo-cis-14-eicosenoic acid-   12-oxo-cis-14,cis-17-eicosadienoic acid-   15-oxo-cis-11,cis-17-eicosadienoic acid-   15-oxo-cis-8,cis-11-eicosadienoic acid-   12-oxo-cis-8,cis-14-eicosadienoic acid-   12-oxo-cis-5,cis-8-eicosadienoic acid-   12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid-   15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid-   15-oxo-cis-5,cis-11-eicosadienoic acid-   15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid-   10,13-dioxo-cis-6-octadecenoic acid-   10,13-dioxo-cis-15-octadecenoic acid-   10,13-dioxo-cis-6,cis-15-octadecadienoic acid-   14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid

Since the hydroxylated fatty acids and oxo fatty acids obtained by thepresent invention have an activity as an agonist of nuclear receptorsPPARα and PPARγ, they can be utilized as metabolism improving agents andthe like. Therefore, novel hydroxylated fatty acids and oxo fatty acidsare also novel substances that can be utilized as metabolism improvingagents and the like.

The hydroxylated fatty acid and oxo fatty acid obtained in the presentinvention can be used by being blended with, for example, medicament,food or cosmetic agent based on the conventionally-known physiologicalactivity.

The dosage form of the medicaments containing hydroxylated fatty acid,oxo fatty acid includes, for example, powder, granule, pill, softcapsule, hard capsules, tablet, chewable tablet, quick-integratingtablet, syrup, liquid, suspension, suppository, ointment, cream, gel,adhesive, inhalant, injection and the like. A preparation thereof isformulated according to a conventional method. Since oxo fatty acid andthe like are poorly soluble in water, they are dissolved in anon-hydrophilic organic solvent such as plant-derived oil,animal-derived oil and the like or dispersed or emulsified in an aqueoussolution together with an emulsifier, a dispersing agent, a surfactantand the like by a homogenizer (high-pressure homogenizer) and used.

Examples of the additives that can be used for formulating includeanimal and plant oils such as soybean oil, safflower oil, olive oil,germ oil, sunflower oil, beef fat, sardine oil and the like,polyalcohols such as polyethylene glycol, propylene glycol, glycerol,sorbitol and the like, surfactants such as sorbitan ester of fatty acid,sucrose ester of fatty acid, glycerin fatty acid ester, polyglycerolester of fatty acid and the like, excipients such as purified water,lactose, starch, crystalline cellulose, D-mannitol, lecithin, gumarabic, sorbitol solution, carbohydrate solution and the like,sweetener, colorant, pH adjuster, flavor and the like. A liquidpreparation may be dissolved or suspended in water or other suitablemedium when in use. Also, tablet and granules may be coated by awell-known method.

For administration in the form of an injection, intravenous,intraperitoneal, intramuscular, subcutaneous, transdermal,intraarticular, intrasynovial, intrathecal, intraperiosteum, sublingual,oral administrations and the like are preferable, and intravenousadministration or intraperitoneal administration is particularlypreferable. The intravenous administration may be any of dripadministration and bolus administration.

Examples of the form of the “food” containing the hydroxylated fattyacid, oxo fatty acid and the like obtained by the present inventioninclude supplement (powder, granule, soft capsule, hard capsules,tablet, chewable tablet, quick-integrating tablet, syrup, liquid etc.),drinks (tea drinks, carbonic acid drinks, lactic acid drinks, sportdrinks etc.), confectionery (gummy candy, jelly, gum, chocolate, cookie,candy etc.), oil, fat and oil foods (mayonnaise, dressing, butter,cream, margarine etc.) and the like.

The above-mentioned foods can contain, where necessary, variousnutrients, various vitamins (vitamin A, vitamin B1, vitamin B2, vitaminB6, vitamin C, vitamin D, vitamin E, vitamin K etc.), various minerals(magnesium, zinc, iron, sodium, potassium, selenium etc.), dietaryfiber, dispersing agent, stabilizer such as emulsifier and the like,sweetener, flavor components (citric acid, malic acid etc.), flavor,royal jelly, propolis, Agaricus and the like.

Examples of the “cosmetics” containing the hydroxylated fatty acid, oxofatty acid and the like obtained by the present invention include cream,skin milk, toner, microemulsion essence, bathing powder and the like,and flavor and the like may be added.

The present invention is explained in more detail in the following byreferring to Examples. The Examples are mere exemplifications of thepresent invention and do not limit the scope of the present invention inany manner.

Example 1 Culture Method of Lactobacillus acidophilus

Lactobacillus acidophilus was inoculated in 15 ml of MRS liquid medium(manufactured by Difco; pH 6.5) from MRS high layer medium containing 2%agar and preserved at 4° C., and cultured at 37° C. for 20 hr. After theculture, the cells were collected by centrifugation at 3,000 rpm, 4° C.for 10 min to give the fungus of Lactobacillus acidophilus.

The aforementioned Lactobacillus acidophilus was deposited on Jan. 17,2014 at the NITE Patent Microorganisms Depositary (NPMD) under depositnumber NITE BP-01788.

Example 2 Cloning of Gene of Enzyme (FA-HY: Fatty Acid Hydration Enzyme)Having the Amino Acid Sequence Shown in SEQ ID NO: 2 (1) Obtainment ofGenome DNA

The aforementioned Lactobacillus acidophilus was inoculated in 10 ml ofMRS liquid medium (manufactured by Difco) and standing culture wasperformed at 37° C. overnight, and the cells were collected bycentrifugation. The obtained fungus was washed twice with 1 ml ofsterile water and added with 0.1 ml of sterile water, 0.125 ml of lysisbuffer (80 mM EDTA containing 200 mM Tris-HCl buffer (pH 8.0)), 0.25 mlof TE saturated phenol, and the mixture was vortexed for 1 min and thesupernatant was recovered by centrifugation. To the obtained supernatant(0.2 ml) was added 0.2 ml of PCI solution (manufactured by NacalaiTesque), and the mixture was hand-shaken, and the supernatant (0.125 ml)was recovered by centrifugation at 15,000 rpm, 4° C., 5 min. 0.0125 mlof 5 M NaCl, 0.31 ml of ethanol were added, and the mixture was stood atroom temperature for 10 min and centrifuged at 15,000 rpm, 4° C., for 5min. The supernatant was discarded and the pellets were washed with 0.2ml of 70% ethanol. 0.1 ml of sterile water was added to the pellets andthe mixture was incubated at 65° C. for 10 min to give genomic DNA.

(2) Obtainment of FA-HY Gene by PCR

In the published total genomic gene sequence of Lactobacillusacidophilus strain, open-reading-frame (ORF) having homology (about 32%)with gene sequence of CLA-HY in WO/2013/168310 was targeted. A senseprimer (SEQ ID NO: 3) was designed based on the sequence of the 5′ sideof the initiation codon of ORF, and an antisense primer (SEQ ID NO: 4)was designed based on the sequence of the 3′ side of the stop codon.Using these primers, genome DNA of Lactobacillus acidophilus as atemplates, PCR was performed. The base sequence of about 1.8 kbp genesegment amplified as a result of PCR was analyzed. It was clarified thatthis gene segment contained a 1,773 bp one open-reading-frame (ORF) (SEQID NO: 1) starting from the initiation codon ATG and ending at stopcodon TAG, and this gene was taken as FA-HY gene. The FA-HY gene encodesa protein consisting of 590 residual amino acids shown in SEQ ID NO: 2.

Example 3 Expression of (FA-HY) in Escherichia coli

A host vector system consisting of Escherichia coli expression vectorpET21b (Novagen) and Rosetta 2 (DE3) strain was used. FA-HY gene segmentamplified by PCR using genomic DNA of Lactobacillus acidophilus as atemplate was inserted into pET21b to construct an expression vector(pFA-HY). Rosetta 2 (DE3) strain was transformed with pFA-HY to give atransformed Rosetta/pFA-HY strain. The obtained Rosetta/pFA-HY strainwas aerobically cultured at 37° C., 300 rpm in a 10 ml LB mediumcontaining 0.5 mg ampicillin, 0.3 mg chloramphenicol (medium containing1% Bacto Tripton (Difco), 0.5% yeast extract, 1% sodium chloride (pH7.0)), 1 μl of 1 M IPTG was added when OD600 nm was 0.5, and the mixturewas further cultured at 16° C. for 18 hr. After culture, the mixture wascentrifuged at 3,000 rpm for 10 min to give wet fungus of Rosetta/pFA-HYstrain.

Example 4 Production of Hydroxylated Fatty Acid from Unsaturated FattyAcid Using Transformed Escherichia coli Expressing Fatty Acid HydrationEnzyme

Hydroxylated fatty acid production test from various unsaturated fattyacids was performed using fatty acid hydration enzyme inductiontransformed Escherichia coli. The total amount of the reaction mixturewas adjusted to 1 ml with 100 mM potassium phosphate buffer (pH 6.5)containing fatty acid hydration enzyme induction transformed Escherichiacoli (wet body weight 0.3 g/ml), NADH (5 mM), FAD (0.1 mM), unsaturatedfatty acid (100 mg), BSA (10 mg). The reaction was performed byanaerobically shaking at 37° C., 120 rpm for 16 to 60 hr in the presenceof an oxygen adsorbent Anaeropack (Mitsubishi Chemical Corporation).After the reaction, 2 ml of chloroform, 2 ml of methanol, and 1 ml of0.5% KCl were added to the reaction mixture (1 ml), and the mixture wasstirred and the chloroform layer was recovered. The recovered chloroformlayer was concentrated by a centrifugation evaporator, and the reactionproduct and unreacted substrate were extracted. A part of the extractwas methylesterified and the reaction product was evaluated by gaschromatography.

Example 5 Purification of Resultant Product from Extract Obtained inExample 4 (Mixture Containing Substrate and Resultant Product(Hydroxylated Fatty Acid))

An extract obtained from Example 4 (mixture containing substrate andresultant product (hydroxylated fatty acid)) was subjected tomoderate-pressure chromatography, a solution that came out from thecolumn was recovered in fractions. The recovered each fraction wasanalyzed by LC/MS and gas chromatography, fractions containing resultantproduct (hydroxylated fatty acid) alone were collected and concentratedby a rotary evaporator. A part of the obtained final resultant product(hydroxylated fatty acid) was methylesterified, and the purity of theresultant product was evaluated by gas chromatography. As a result, aresultant product having a purity of not less than 98% was obtained fromeach substrate. The chemical structure of the resultant product wasdetermined by NMR, two-dimensional NMR, GC-MS analysis and the like. Asa result, 10-hydroxy-hexadecanoic acid having a purity of not less than98% could be obtained from cis-9-hexadecenoic acid (pulmitoleic acid).12-Hydroxy-octadecanoic acid having a purity of not less than 98% couldbe obtained from cis-11-octadecenoic acid(cis-vaccenic acid).13-Hydroxy-cis-9-octadecenoic acid having a purity of not less than 98%could be obtained from cis-9,cis-12-octadecadienoic acid (linoleicacid). 13-Hydroxy-cis-6,cis-9-octadecadienoic acid having a purity ofnot less than 98% could be obtained fromcis-6,cis-9,cis-12-octadecatrienoic acid (γ-linolenic acid).13-hydroxy-cis-9,cis-15-octadecadienoic acid having a purity of not lessthan 98% could be obtained from cis-9,cis-12,cis-15-octadecatrienoicacid (α-linolenic acid). 13-Hydroxy-cis-6,cis-9,cis-15-octadecatrienoicacid having a purity of not less than 98% could be obtained fromcis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (stearidonic acid).12-Hydroxy-cis-14-eicosenoic acid and 15-hydroxy-cis-11-eicosenoic acidhaving a purity of not less than 98% could be obtained fromcis-11,cis-14-eicosadienoic acid. 12-Hydroxy-cis-14,cis-17-eicosadienoicacid and 15-hydroxy-cis-11,cis-17-eicosadienoic acid having a purity ofnot less than 98% could be obtained fromcis-11,cis-14,cis-17-eicosatrienoic acid.15-Hydroxy-cis-8,cis-11-eicosadienoic acid and12-hydroxy-cis-8,cis-14-eicosadienoic acid having a purity of not lessthan 98% could be obtained from cis-8,cis-11,cis-14-eicosatrienoic acid(dihomo-γ-linolenic acid). 12-Hydroxy-cis-5,cis-8-eicosadienoic acidhaving a purity of not less than 98% could be obtained fromcis-5,cis-8,cis-11-eicosatrienoic acid (mead acid).12-Hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid and15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid having a purity ofnot less than 98% could be obtained fromcis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid.15-Hydroxy-cis-5,cis-8,cis-11-eicosatrienoic acid and12-hydroxy-cis-5,cis-8,cis-14-eicosatrienoic acid having a purity of notless than 98% could be obtained fromcis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid).10,13-Dihydroxy-octadecanoic acid having a purity of not less than 98%could be obtained from 10-hydroxy-cis-12-octadecenoic acid.10,13-Dihydroxy-cis-6-octadecenoic acid having a purity of not less than98% could be obtained from 10-hydroxy-cis-6, cis-12-octadecadienoicacid. 10,13-Dihydroxy-cis-15-octadecenoic acid having a purity of notless than 98% could be obtained from10-hydroxy-cis-12,cis-15-octadecadienoic acid.10,13-Dihydroxy-cis-6,cis-15-octadecadienoic acid having a purity of notless than 98% could be obtained from10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid.10-Oxo-13-hydroxy-octadecanoic acid having a purity of not less than 98%could be obtained from 10-oxo-cis-12-octadecenoic acid.10-Oxo-13-hydroxy-cis-6-octadecenoic acid having a purity of not lessthan 98% could be obtained from 10-oxo-cis-6,cis-12-octadecadienoicacid. 10-Oxo-13-hydroxy-cis-15-octadecenoic acid having a purity of notless than 98% could be obtained from10-oxo-cis-12,cis-15-octadecadienoic acid.10-Oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid having a purity ofnot less than 98% could be obtained from10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid.13-Hydroxy-cis-5,cis-9-octadecadienoic acid having a purity of not lessthan 98% could be obtained from cis-5,cis-9,cis-12-octadecatrienoic acid(pinolenic acid). 13-Hydroxy-trans-5,cis-9-octadecadienoic acid having apurity of not less than 98% could be obtained fromtrans-5,cis-9,cis-12-octadecatrienoic acid (columbinic acid).15-Hydroxy-cis-5,cis-11-eicosadienoic acid having a purity of not lessthan 98% could be obtained from cis-5,cis-11,cis-14-eicosatrienoic(Sciadonic acid). 15-Hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acidhaving a purity of not less than 98% could be obtained fromcis-5,cis-11,cis-14,cis-17-eicosatetraenoic acid (juniperonic acid).14-Hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid havinga purity of not less than 98% could be obtained fromcis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid (DHA).10-Hydroxy-tetradecanoic acid having a purity of not less than 98% couldbe obtained from cis-9-tetradecenoic acid(myristoleic acid).

Example 6 Production of Oxo Fatty Acid from Hydroxylated Fatty Acid byUsing Dehydrogenase (CLA-DH) Expressed in Escherichia coli

An oxo fatty acid production test from hydroxylated fatty acid wasperformed using purified dehydrogenase (CLA-DH) derived from L.plantarum FERN BP-10549 strain obtained by the method described inJP-A-2007-259712 or the method described in WO/2013/168310. The reactionmixture was adjusted to the total amount of 1 ml with 50 mM potassiumphosphate buffer (pH 8.0) containing purified dehydrogenase (enzymeamount 83 μg), 0.5 mM NAD⁺, 0.5 mg hydroxylated fatty acid. The reactionwas performed by anaerobically shaking at 37° C., 120 rpm for 24 hr inthe presence of an oxygen adsorbent Anaeropack (Mitsubishi ChemicalCorporation). After the reaction, lipid was extracted by Bligh-Dyermethod from the reaction mixture. The extract was methylesterified andproduction of oxo fatty acid was evaluated by gas chromatography. As aresult, production of oxo fatty acid 13-oxo-cis-9-octadecenoic acid(0.01 mg) was confirmed when hydroxylated fatty acid13-hydroxy-cis-9-octadecenoic acid was used as a substrate. Productionof 13-oxo-cis-9,cis-15-octadecadienoic acid (0.02 mg) was confirmed when13-hydroxy-cis-9,cis-15-octadecadienoic acid was used as a substrate,and production of 13-oxo-cis-6,cis-9-octadecadienoic acid (0.06 mg) wasconfirmed when 13-hydroxy-cis-6,cis-9-octadecadienoic acid was used as asubstrate.

Using a similar method, the resultant product oxo fatty acid can beobtained by using each hydroxylated fatty acid as a substrate.

Example 7 Production of Oxo Fatty Acid from Hydroxylated Fatty Acid byUsing Anhydrous Chromic Acid (CrO₃)

To anhydrous chromic acid (2.67 g) were added sulfuric acid (2.3 ml) andwater (7.7 ml), and acetone (90 ml) was added thereto to give a chromicacid solution. 2 g of hydroxylated fatty acid and 40 ml of acetone wereadded into an Erlenmeyer flask, and the above-mentioned chromic acidsolution was added drop by drop on ice while stirring the mixture with astirrer. When the solution turned from blue to the color of powderedgreen tea, the dropwise addition of the chromic acid solution wasstopped and the reaction was quenched with isopropyl alcohol. Theprecipitated sediment was filtered with a filter paper and placed in apartitioning funnel. Diethyl ether (150 ml) and Milli Q water (300 ml)were further added and the mixture was shaken well. The diethyl etherlayer was washed several times with Milli Q water. To the diethyl etherlayer after washing was added an appropriate amount of sodium sulfate(anhydrous), the mixture was stirred, and the residual water wasremoved. The anhydrous sodium sulfate added was filtered off with afilter paper, the obtained diethyl ether layer was concentrated in arotary evaporator, and the reaction product (oxo fatty acid) and anunreacted substrate were extracted.

Example 8 Purification of Resultant Product from Extracts (MixtureContaining Substrate and Resultant Product (Oxo Fatty Acid)) Obtained inExamples 6, 7

The extracts (mixture containing substrate and resultant product (oxofatty acid)) obtained in Examples 6, 7 were subjected tomoderate-pressure chromatography, a solution that came out from thecolumn was recovered in fractions. The recovered each fraction wasanalyzed by LC/MS and gas chromatography, fractions containinghydroxylated fatty acid alone were collected and concentrated by arotary evaporator. A part of the obtained final resultant product wasmethylesterified, and the purity of oxo fatty acid was evaluated by gaschromatography. As a result, oxo fatty acid having a purity of not lessthan 98% was obtained from each substrate. The chemical structure of theresultant product was determined by NMR, two-dimensional NMR, GC-MSanalysis and the like. As a result, 13-oxo-cis-9-octadecenoic acidhaving a purity of not less than 98% could be obtained from13-hydroxy-cis-9-octadecenoic acid. 13-Oxo-cis-6,cis-9-octadecadienoicacid having a purity of not less than 98% could be obtained from13-hydroxy-cis-6, cis-9-octadecadienoic acid.13-Oxo-cis-9,cis-15-octadecadienoic acid having a purity of not lessthan 98% could be obtained from 13-hydroxy-cis-9,cis-15-octadecadienoicacid.

Using a similar method, 10-oxo-hexadecanoic acid having a purity of notless than 98% can be obtained from 10-hydroxy-hexadecanoic acid.12-Oxo-octadecanoic acid having a purity of not less than 98% can beobtained from 12-hydroxy-octadecanoic acid.13-Oxo-cis-6,cis-9,cis-15-octadecatrienoic acid having a purity of notless than 98% can be obtained from13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid.15-Oxo-cis-11-eicosenoic acid having a purity of not less than 98% canbe obtained from 15-hydroxy-cis-11-eicosenoic acid.12-Oxo-cis-14-eicosenoic acid having a purity of not less than 98% canbe obtained from 12-hydroxy-cis-14-eicosenoic acid.12-Oxo-cis-14,cis-17-eicosadienoic acid having a purity of not less than98% can be obtained from 12-hydroxy-cis-14,cis-17-eicosadienoic acid.15-Oxo-cis-11,cis-17-eicosadienoic acid having a purity of not less than98% can be obtained from 15-hydroxy-cis-11,cis-17-eicosadienoic acid.15-Oxo-cis-8,cis-11-eicosadienoic acid having a purity of not less than98% can be obtained from 15-hydroxy-cis-8,cis-11-eicosadienoic acid.12-Oxo-cis-8,cis-14-eicosadienoic acid having a purity of not less than98% can be obtained from 12-hydroxy-cis-8,cis-14-eicosadienoic acid.12-Oxo-cis-5,cis-8-eicosadienoic acid having a purity of not less than98% can be obtained from 12-hydroxy-cis-5,cis-8-eicosadienoic acid.12-Oxo-cis-8,cis-14,cis-17-eicosatrienoic acid having a purity of notless than 98% can be obtained from12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid.15-Oxo-cis-8,cis-11,cis-17-eicosatrienoic acid having a purity of notless than 98% can be obtained from15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid.15-Oxo-cis-5,cis-8,cis-11-eicosatrienoic acid having a purity of notless than 98% can be obtained from15-hydroxy-cis-5,cis-8,cis-11-eicosatrienoic acid.12-Oxo-cis-5,cis-8,cis-14-eicosatrienoic acid having a purity of notless than 98% can be obtained from12-hydroxy-cis-5,cis-8,cis-14-eicosatrienoic acid.10,13-Dioxo-octadecanoic acid having a purity of not less than 98% canbe obtained from 10,13-dihydroxy-octadecanoic acid or10-oxo-13-hydroxy-octadecanoic acid. 10,13-Dioxo-cis-6-octadecenoic acidhaving a purity of not less than 98% can be obtained from10,13-dihydroxy-cis-6-octadecenoic acid or10-oxo-13-hydroxy-cis-6-octadecenoic acid.10,13-Dioxo-cis-15-octadecenoic acid having a purity of not less than98% can be obtained from 10,13-dihydroxy-cis-15-octadecenoic acid or10-oxo-13-hydroxy-cis-15-octadecenoic acid.10,13-Dioxo-cis-6,cis-15-octadecadienoic acid having a purity of notless than 98% can be obtained from10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid or10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid. 13-Oxo-cis-5,cis-9-octadecadienoic acid having a purity of not less than 98% can beobtained from 13-hydroxy-cis-5, cis-9-octadecadienoic acid.13-Oxo-trans-5, cis-9-octadecadienoic acid having a purity of not lessthan 98% can be obtained from 13-hydroxy-trans-5,cis-9-octadecadienoicacid. 15-Oxo-cis-5,cis-11-eicosadienoic acid having a purity of not lessthan 98% can be obtained from 15-hydroxy-cis-5,cis-11-eicosadienoicacid. 15-Oxo-cis-5,cis-11,cis-17-eicosatrienoic acid having a purity ofnot less than 98% can be obtained from15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid.14-Oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid having apurity of not less than 98% can be obtained from14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid.10-Oxo-tetradecanoic acid having a purity of not less than 98% can beobtained from 10-hydroxy-tetradecanoic acid.

Example 9 Measurement of Activity as Agonist of Nuclear Receptors PPARαand PPARγ

The PPARα,γ activation ability of the PPAR ligand of the presentinvention was measured in reference to Nobuyuki Takahashi et al., FEBSLetters 514 (2002) p. 315-322, “Dual action of isoprenols from herbalmedicines on both PPARgamma and PPARalpha in 3T3-L1 adipocytes and HepG2hepatocytes.”, the section of Material and Methods “Reporter plasmidsand luciferase assays”. To be specific, PPARα,γ ligand activity wasmeasured by a reporter assay that evaluates binding to a fusion proteinof PPAR ligand binding region and GAL4 DNA binding region and targetgene activation, based on the expression of luciferase. Specifically, aplasmid comprising a DNA encoding a fusion protein of PPARα,γ ligandbinding region and GAL4 DNA binding region and a reporter plasmidwherein luciferase linked to GAL4 bound DNA sequence were introducedinto CV-1 cell, a ligand described below was added to the cell, the cellwas incubated and the luciferase activity was detected.

The concentration of the sample was adjusted with ethanol. Ethanol wasused as a negative control, and PPARα,γ ligands GW7647 (10 nM) andtroglitazone (5 μM) were used as a positive control.

The PPARα, γ ligand activity data of representative hydroxylated fattyacid, oxo fatty acid, (1) 13-hydroxy-cis-9-octadecenoic acid (indicatedas “13-OH LA”), (2) 13-oxo-cis-9-octadecenoic acid (indicated as “13-OXOLA”), (3) 10,13-dihydroxy-octadecanoic acid (indicated as “10,13-OHLA”), (4) 13-hydroxy-cis-9,cis-15-octadecadienoic acid (indicated as“13-OH ALA”), (5) 13-oxo-cis-9,cis-15-octadecadienoic acid (indicated as“13-OXO ALA”), (6) 10,13-dihydroxy-cis-15-octadecenoic acid (indicatedas “10,13-OH ALA”) (7) 13-hydroxy-cis-6,cis-9-octadecadienoic acid(indicated as “13-OH GLA”), (8) 13-oxo-cis-6,cis-9-octadecadienoic acid(indicated as “13-OXO GLA”), (9) 10,13-dihydroxy-cis-6-octadecenoic acid(indicated as “10,13-OH GLA”), (10)15-hydroxy-cis-5,cis-8,cis-11-eicosatrienoic acid (indicated as “15-OHAA”) are shown in FIG. 1 and FIG. 2. Of these, rare fatty acids of (4),(6), (8) and (9) are novel fatty acids.

While the present invention has been described with emphasis onpreferred embodiments, it is obvious to those skilled in the art thatthe preferred embodiments can be modified.

The contents disclosed in any publication cited herein, includingpatents and patent applications, are hereby incorporated in theirentireties by reference, to the extent that they have been disclosedherein.

INDUSTRIAL APPLICABILITY

According to the method of the present invention, various hydroxylatedfatty acids and oxo fatty acids can be produced, and therefore, thehydroxylated fatty acids and oxo fatty acids can be applied to variousfield of medicament, food and the like. In addition, according to themethod of the present invention, novel rare fatty acid can be produced,which is extremely useful.

This application is based on a patent application No. 2014-011855 filedin Japan (filing date: Jan. 24, 2014), the contents of which areincorporated in full herein.

1. An enzyme protein of any of the following (a)-(c): (a) the enzymeprotein consisting of the amino acid sequence shown in SEQ ID NO: 2, (b)a protein comprising an amino acid sequence wherein one or plural aminoacids in the amino acid sequence shown in SEQ ID NO: 2 are deletedand/or substituted and/or inserted and/or added, and having an enzymeactivity that catalyzes a hydration reaction, (c) a protein encoded by abase sequence that hybridizes to a nucleic acid consisting of a chainsequence complementary to the base sequence shown in SEQ ID NO: 1 understringent conditions, and having an enzyme activity that catalyzes ahydration reaction.
 2. Lactobacillus acidophilus fungus or fungal debristhereof, comprising the enzyme protein according to claim
 1. 3. Anucleic acid encoding the enzyme protein according to claim
 1. 4. Avector comprising the nucleic acid according to claim
 3. 5. A host celltransformed with the vector according to claim
 4. 6. A method ofproducing the enzyme protein according to claim 1, comprising culturingthe host cell according to claim 5 and recovering the enzyme from theculture.
 7. A method of producing a hydroxylated fatty acid having 18carbon atoms and a hydroxyl group at the 13-position, comprisingsubjecting an unsaturated fatty acid having 18 carbon atoms and a cisdouble bond at the 12-position to a hydration reaction using the enzymeprotein according to claim 1, wherein the unsaturated fatty acid having18 carbon atoms and a cis double bond at the 12-position is linoleicacid, γ-linolenic acid, α-linolenic acid, stearidonic acid,10-hydroxy-cis-12-octadecenoic acid,10-hydroxy-cis-6,cis-12-octadecadienoic acid,10-hydroxy-cis-12,cis-15-octadecadienoic acid,10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid,10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoicacid, 10-oxo-cis-12,cis-15-octadecadienoic acid,10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, pinolenic acid, orcolumbinic acid.
 8. A method of producing an oxo fatty acid having 18carbon atoms and a carbonyl group at the 13-position, comprisingsubjecting an unsaturated fatty acid having 18 carbon atoms and a cisdouble bond at the 12-position to a hydration reaction using the enzymeprotein according to claim 1 to induce a hydroxylated fatty acid having18 carbon atoms and a hydroxyl group at the 13-position, and subjectingthe hydroxylated fatty acid to a dehydrogenation reaction or chemicaloxidation, wherein the unsaturated fatty acid having 18 carbon atoms anda cis double bond at the 12-position is linoleic acid, γ-linolenic acid,α-linolenic acid, stearidonic acid, 10-hydroxy-cis-12-octadecenoic acid,10-hydroxy-cis-6,cis-12-octadecadienoic acid,10-hydroxy-cis-12,cis-15-octadecadienoic acid,10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid,10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoicacid, 10-oxo-cis-12,cis-15-octadecadienoic acid,10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, pinolenic acid, orcolumbinic acid, and wherein the dehydrogenation reaction uses adehydrogenase derived from lactobacillus.
 9. A method of producing ahydroxylated fatty acid having 16 carbon atoms and a hydroxyl group atthe 10-position, comprising subjecting an unsaturated fatty acid having16 carbon atoms and a cis double bond at the 9-position to a hydrationreaction using the enzyme protein according to claim 1, wherein theunsaturated fatty acid having 16 carbon atoms and a cis double bond atthe 9-position is pulmitoleic acid.
 10. A method of producing an oxofatty acid having 16 carbon atoms and a carbonyl group at the10-position, comprising subjecting an unsaturated fatty acid having 16carbon atoms and a cis double bond at the 9-position to a hydrationreaction using the enzyme protein according to claim 1 to induce ahydroxylated fatty acid having 16 carbon atoms and a hydroxyl group atthe 10-position, and subjecting the hydroxylated fatty acid to adehydrogenation reaction or chemical oxidation, wherein the unsaturatedfatty acid having 16 carbon atoms and a cis double bond at the9-position is pulmitoleic acid, and wherein the dehydrogenation reactionuses a dehydrogenase derived from lactobacillus.
 11. A method ofproducing a hydroxylated fatty acid having 20 carbon atoms and ahydroxyl group at the 15-position, comprising subjecting an unsaturatedfatty acid having 20 carbon atoms and a cis double bond at the14-position to a hydration reaction using the enzyme protein accordingto claim 1, wherein the unsaturated fatty acid having 20 carbon atomsand a cis double bond at the 14-position is cis-11,cis-14-eicosadienoicacid, cis-11,cis-14,cis-17-eicosatrienoic acid, dihomo-γ-linolenic acid,cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, arachidonic acid,sciadonic acid, or juniperonic acid.
 12. A method of producing an oxofatty acid having 20 carbon atoms and a carbonyl group at the15-position, comprising subjecting an unsaturated fatty acid having 20carbon atoms and a cis double bond at the 14-position to a hydrationreaction using the enzyme protein according to claim 1 to induce ahydroxylated fatty acid having 20 carbon atoms and a hydroxyl group atthe 15-position, and subjecting the hydroxylated fatty acid to adehydrogenation reaction or chemical oxidation, wherein the unsaturatedfatty acid having 20 carbon atoms and a cis double bond at the14-position is cis-11,cis-14-eicosadienoic acid,cis-11,cis-14,cis-17-eicosatrienoic acid, dihomo-γ-linolenic acid,cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, arachidonic acid,sciadonic acid, or juniperonic acid, and wherein the dehydrogenationreaction uses a dehydrogenase derived from lactobacillus.
 13. A methodof producing a hydroxylated fatty acid having 18 or 20 carbon atoms anda hydroxyl group at the 12-position, comprising subjecting anunsaturated fatty acid having 18 or 20 carbon atoms and a cis doublebond at the 11-position to a hydration reaction using the enzyme proteinaccording to claim 1, wherein the unsaturated fatty acid having 18 or 20carbon atoms and a cis double bond at the 11-position is cis-vaccenicacid, cis-11-cis-14-eicosadienoic acid,cis-11,cis-14,cis-17-eicosatrienoic, dihomo-γ-linolenic acid, mead acid,cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, or arachidonic acid.14. A method of producing an oxo fatty acid having 18 or carbon atomsand a carbonyl group at the 12-position, comprising subjecting anunsaturated fatty acid having 18 or 20 carbon atoms and a cis doublebond at the 11-position to a hydration reaction using the enzyme proteinaccording to claim 1 to induce a hydroxylated fatty acid having 18 or 20carbon atoms and a hydroxyl group at the 12-position, and subjecting thehydroxylated fatty acid to a dehydrogenation reaction or chemicaloxidation, wherein the unsaturated fatty acid having 18 or 20 carbonatoms and a cis double bond at the 11-position is cis-vaccenic acid,cis-11-cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic,dihomo-γ-linolenic acid, mead acid,cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, or arachidonic acid,and wherein the dehydrogenation reaction uses a dehydrogenase derivedfrom lactobacillus. 15.-18. (canceled)
 19. A method of producing14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid,comprising subjectingcis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to ahydration reaction using the enzyme protein according to claim
 1. 20. Amethod of producing14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid,comprising subjectingcis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to ahydration reaction using the enzyme protein according to claim 1 toinduce 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoicacid, and subjecting the hydroxylated fatty acid to a dehydrogenationreaction or chemical oxidation, wherein the dehydrogenation reactionuses a dehydrogenase derived from lactobacillus.
 21. A method ofproducing 10-hydroxy-tetradecanoic acid, comprising subjectingcis-9-tetradecenoic acid (myristoleic acid) to a hydration reactionusing the enzyme protein according to claim
 1. 22. A method of producing10-oxo-tetradecanoic acid, comprising subjecting cis-9-tetradecenoicacid (myristoleic acid) to a hydration reaction using the enzyme proteinaccording to claim 1 to induce 10-hydroxy-tetradecanoic acid, andsubjecting the hydroxylated fatty acid to a dehydrogenation reaction orchemical oxidation, wherein the dehydrogenation reaction uses adehydrogenase derived from lactobacillus. 23.-24. (canceled) 25.13-Hydroxy-cis-9,cis-15-octadecadienoic acid,13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid,10,13-dihydroxy-cis-6-octadecenoic acid,10,13-dihydroxy-cis-15-octadecenoic acid,10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid,10-oxo-13-hydroxy-cis-6-octadecenoic acid,10-oxo-13-hydroxy-cis-15-octadecenoic acid,10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid,13-hydroxy-cis-5,cis-9-octadecadienoic acid, or13-hydroxy-trans-5,cis-9-octadecadienoic acid, which is produced by themethod according to claim
 7. 26. 15-Hydroxy-cis-11,cis-17-eicosadienoicacid, 15-hydroxy-cis-8, cis-11-eicosadienoic acid,15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid,15-hydroxy-cis-5,cis-11-eicosadienoic acid, or15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced bythe method according to claim
 11. 27. 12-Hydroxy-cis-14-eicosenoic acid,12-hydroxy-cis-14,cis-17-eicosadienoic acid or12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid, which is produced bythe method according to claim
 13. 28. 13-Oxo-cis-6,cis-9-octadecadienoicacid, 13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid,10,13-dioxo-cis-6-octadecenoic acid, 10,13-dioxo-cis-15-octadecenoicacid, 10,13-dioxo-cis-6,cis-15-octadecadienoic acid,13-oxo-cis-5,cis-9-octadecadienoic acid or13-oxo-trans-5,cis-9-octadecadienoic acid, which is produced by themethod according to claim
 8. 29. 15-Oxo-cis-11,cis-17-eicosadienoicacid, 15-oxo-cis-8,cis-11-eicosadienoic acid,15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid,15-oxo-cis-5,cis-11-eicosadienoic acid or15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced by themethod according to claim
 12. 30. 12-Oxo-cis-14-eicosenoic acid,12-oxo-cis-14,cis-17-eicosadienoic acid,12-oxo-cis-8,cis-14-eicosadienoic acid, 12-oxo-cis-5,cis-8-eicosadienoicacid or 12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid, which isproduced by the method according to claim
 14. 31.14-Hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acidproduced by the method according to claim
 19. 32.14-Oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, which isproduced by the method according to claim
 20. 33. A fattyacid-containing substance comprising13-hydroxy-cis-9,cis-15-octadecadienoic acid,13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid,10,13-dihydroxy-cis-6-octadecenoic acid,10,13-dihydroxy-cis-15-octadecenoic acid,10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid,10-oxo-13-hydroxy-cis-6-octadecenoic acid,10-oxo-13-hydroxy-cis-15-octadecenoic acid,10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid,13-hydroxy-cis-5,cis-9-octadecadienoic acid,13-hydroxy-trans-5,cis-9-octadecadienoic acid,15-hydroxy-cis-11,cis-17-eicosadienoic acid,15-hydroxy-cis-8,cis-11-eicosadienoic acid,15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid,15-hydroxy-cis-5,cis-11-eicosadienoic acid,15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid,12-hydroxy-cis-14-eicosenoic acid,12-hydroxy-cis-14,cis-17-eicosadienoic acid,12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid,13-oxo-cis-6,cis-9-octadecadienoic acid,13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid,10,13-dioxo-cis-6-octadecenoic acid, 10,13-dioxo-cis-15-octadecenoicacid, 10,13-dioxo-cis-6,cis-15-octadecadienoic acid,13-oxo-cis-5,cis-9-octadecadienoic acid,13-oxo-trans-5,cis-9-octadecadienoic acid,15-oxo-cis-11,cis-17-eicosadienoic acid,15-oxo-cis-8,cis-11-eicosadienoic acid,15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid,15-oxo-cis-5,cis-11-eicosadienoic acid,15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid, 12-oxo-cis-14-eicosenoicacid, 12-oxo-cis-14,cis-17-eicosadienoic acid,12-oxo-cis-8,cis-14-eicosadienoic acid, 12-oxo-cis-5,cis-8-eicosadienoicacid, 12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid,14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, or14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid. 34.(canceled)